Objective It was previously reported that docosahexanoic acid (DHA) reduces TNF-α-induced necrosis in L929 cells. However, the mechanisms underlying this reduction have not been investigated. The present study was designed to investigate cellular and biochemical mechanisms underlying the attenuation of TNF-α-induced necroptosis by DHA in L929 cells. Methods L929 cells were pre-treated with DHA prior to exposure to TNF-α, zVAD, or Necrostatin-1 (Nec-1). Cell death and survival were assessed by MTT and caspase activity assays, and microscopic visualization. Reactive oxygen species (ROS) were measured by flow cytometry. C16- and C18-ceramide were measured by mass spectrometry. Lysosomal membrane permeabilization (LMP) was evaluated by fluorescence microscopy and flow cytometry using Acridine Orange. Cathepsin L activation was evaluated by immunoblotting and fluorescence microscopy. Autophagy was assessed by immunoblotting of LC3-II and Beclin. Results Exposure of L929 cells to TNF-α alone for 24 h induced necroptosis, as evidenced by inhibition of cell death by Nec-1, absence of caspase-3 activity and lamin B cleavage, and morphological analysis. DHA attenuated multiple biochemical events associated with TNF-α-induced necroptosis, including ROS generation, ceramide production, lysosomal dysfunction, cathepsin L activation, and autophagic features. DHA also attenuated zVAD-induced necroptosis but did not attenuate the enhanced apoptosis and necrosis induced by combination of TNF-α with Actinomycin D or zVAD, respectively, suggesting that its protective effects might be limited by the strength of the cell death insult induced by TNF-α. Conclusions DHA effectively attenuates TNF-α-induced necroptosis and autophagy, most likely via its ability to inhibit TNF-α-induced sphingolipid metabolism and oxidative stress. These results highlight the role of this Omega-3 fatty acid in antagonizing inflammatory cell death.

High-risk types of human papillomavirus (HPV) cause nearly all cases of cervical cancer. The E6 oncoprotein is produced as a full-length variant (E6) as well as several shorter isoforms (E6*). E6* inhibits certain oncogenic activities of E6, suggesting that it might play an anti-oncogenic role in vivo. To test this, we created E6*-expressing SiHa (HPV+) and C33A (HPV−) cells, then examined the ability of both the parental and E6*-expressing cells to form tumors in nude mice. We found that over-expression of E6* indeed decreased the growth of tumors derived from both SiHa and C33A cells, with the reduction greatest in tumors derived from E6*-expressing SiHa cells. These findings point to multiple anti-oncogenic characteristics of E6*, some of which likely involve down-regulation of the full-length isoform, and others that are independent of HPV. These data represent the first demonstration of biologically-relevant E6* activities distinct from those of the full-length isoform in vivo.

Additional information is available at the end of the chapter http://dx.doi.org/10.5772/55810 . IntroductionCervical cancer was formerly the second most common cancer killer of women worldwide. Following widespread adoption of Papanicolau cytologic screening Pap test for cervical cancer in the s, this began to change. Today, advanced cervical cancer is rare in screened populations. Although an uncommon disease in developed nations, internationally about , women annually are diagnosed with cervical cancer, and about half of those women will die of their disease. In global terms, this ranks second only to breast cancer as a cause of cancer-specific mortality. Over the past three decades the scientific community has witnessed spectacular advances in the understanding of the underlying pathophysiology of cervical cancer, with the most profound discovery being in of the identification of the human papillomavirus HPV within cervical cancer a discovery that earned Harold Zur-Hausen, M.D the Nobel prize for Medicine and Physiology in . A viral etiology for cervical cancer implied that it may be possible to eradicate cervical cancer through vaccination. This promise was partially fulfilled in when the United States Food and Drug Administration approved an HPV vaccine for the prevention of HPV-induced cervical dysplasia and/or cancer. These advances, profound though they are, have yet to eradicate cervical cancer. Furthermore, due to the pervasiveness of HPV infection and the timeline of disease progression, it will be a few decades before we will be able to determine the impact preventive practices are having on cancer incidence and prevalence. In addition, those for whom preventative measures are not a solution, including HIV + individuals as well as women already infected with HR-HPV, await an answer.Over the past several years, developments in innovative imaging, superior surgical technologies, immunotherapies, and molecular therapies have surfaced, making the eradication of cervical cancer a much more achievable goal than in the past. Several areas of cervical cancer research continue to address the challenges posed by the need for appropriate therapeutic alternatives, and progress is occurring at each level of clinical management ranging from detection to the development of small molecule antiviral leads. Because the field is evolving rapidly in all directions and related disciplines, it is helpful to summarize the status of our growth, and to recognize those pioneering efforts that may ultimately contribute to achieving our goal of eliminating cervical cancer. This review seeks to survey the current understanding of cervical cancer etiology and treatment and to review areas requiring additional progress. . Prevention, interception and early detectionDisease Burden and Risk: Approximately million Americans are currently infected with HPV, and another million new infections occur annually. In about percent of these cases, the infection is cleared by the immune system within two years [ , ]. However, a relatively smal...

High-risk types of the human papillomavirus (HR-HPV) are the causative agents of nearly all cases of cervical cancer, as well as a significant number of head, neck, penile, vulvar and anal cancers. Like many other viruses with small genomes, HPV (∼8 kb) utilizes numerous mechanisms to increase the capacity of its genome to encode the proteins necessary for successful completion of its infectious life cycle, including alternative splicing. Studies over the past few decades have focused intensively on the activities and roles of E6 proteins from HR-HPVs during the process of cellular transformation, clearly implicating E6 as a major transforming agent. In contrast, the role of the smaller splice isoform, E6*, in the carcinogenic process has not yet been established. In a recent study, we demonstrated that the over-expression of E6* reduces tumor growth by SiHa (HPV16 positive) and C33A (no HPV) cells in nude mice, suggesting that therapies emulating the actions of E6* may be of medical benefit. Furthermore, tumor growth inhibition by E6* was greater in tumors derived from HPV positive cells than in tumors derived from HPV negative cells. This difference implies that E6* interferes with the oncogenic activity of the full-length protein as well as by acting through HPV-independent mechanisms. The goal of this study is to determine the pathways affected by E6* that may lead to the observed reduction in tumor formation in xenograft models. To elucidate how E6* may affect the levels of cellular proteins and thereby orchestrate pathway regulation, in both E6 positive and negative environments, SiHa pFlag, SiHa pE6*, C33A pFlag, and C33A pE6* cells were created and their differential protein expression examined using mass spectrometry and Ingenuity Pathway Analysis (IPA) software. Lysates of these cells were reduced, alkylated, trypsinized, and TMT labeled, and the labeled peptides were analyzed using an LTQ-Orbitrap Velos mass spectrometer. Proteins were quantified by TMT tags and identified by comparison against the human library using Proteome Discoverer Software. 322 proteins were detected as differentially expressed using a 1.3 fold-change cut-off value. Further analysis by IPA revealed that E6* induced changes in apoptosis and death receptor signaling pathways in both HPV- and HPV positive cells, while other pathways, such as those involving mitochondrial dysfunction and TNFR1 signaling, were more profoundly affected in HPV negative cells. Our study provides several promising leads for future experiments and analyses, specifically in the context of human cancers, and carries with it the exciting possibility of replicating the anti-oncogenic activity of E6* in such a way as to provide therapeutic benefit. Future work will involve more detailed examination of our preliminary results and comparing these observations with those obtained from actual tumors derived from these cells. Citation Format: Whitney Evans, Maria Filippova, Robert Aragon, Valeri Filippov, Mark E. Reeves, Penelope Duerksen-Hughes. Proteomic analysis of the effect of E6 star expression on cellular pathways in HPV positive SiHa and HPV negative C33A cervical carcinoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1828. doi:10.1158/1538-7445.AM2015-1828

High-risk types of the human papillomavirus (HPV) are the causative agents of nearly all cases of cervical cancer, as well as a significant number of head, neck, penile, vulvar, and anal cancers. Like many other viruses with small genomes, HPV (∼8 kb) utilizes numerous mechanisms to increase the capacity of its genome to encode the proteins necessary for successful completion of its infectious life cycle, including alternative splicing. Studies over the past few decades have focused intensively on the activities and roles of E6 proteins from high-risk types of HPV during the process of cellular transformation, clearly implicating E6 as a major transforming agent. In contrast, the role of the smaller splice isoform, E6*, in the carcinogenic process (if any) has not yet been established. In the present study, we examined the behavior of the E6* protein during tumor growth in an in vivo nude mouse xenograft model. We created E6*-expressing SiHa (HPV+) and C33A (HPV-) cells, then examined the ability of both the parental and E6*-expressing cells to form tumors in nude mice. The difference in tumor size observed in the presence and absence of E6*, when expressed in both HPV+ (SiHa) and HPV- (C33A) cells, was dramatic and consistent, strongly indicating an anti-oncogenic role for E6* in this context. Interestingly, we found that tumor growth inhibition by E6* was greater in tumors derived from SiHa cells, which are HPV16-positive, than in tumors produced by C33A, which are HPV-negative. This difference implies that E6* acts by interfering with the oncogenic activity of the full-length protein as well as through one or more HPV-independent mechanisms. Consistent with this idea, we found that E6* does indeed bind to the full-sized isoform and inhibits its ability to accelerate degradation of p53 and procaspase 8. These data represent the first demonstration of biologically-relevant E6* activities distinct from those of the full-length isoform in vivo. The significance of these findings in the context of human cancers is in the possibility of mimicking or replicating the anti-oncogenic activity of E6* in such a way as to provide therapeutic benefit. Citation Format: Whitney Evans, Maria Filippova, Robert Aragon, Valeri Filippov, Mark Reeves, Penelope Duerksen-Hughes. The splice variant of the human papillomavirus 16 E6 protein, E6*, displays anti-tumor properties in vivo. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3175. doi:10.1158/1538-7445.AM2014-3175